CN114470162B - Application of GDF6 and over-expression reagent thereof in preparation of myocardial cell protective agent - Google Patents
Application of GDF6 and over-expression reagent thereof in preparation of myocardial cell protective agent Download PDFInfo
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Abstract
The invention relates to the field of biomedicine, and discloses application of GDF6 and an over-expression reagent thereof in preparation of a myocardial cell protective agent. The invention firstly proposes that GDF6 overexpression can reduce the apoptosis and iron death of myocardial cells after myocardial ischemia and promote myocardial survival, and is expected to provide a new theoretical basis and a potential intervention target point for myocardial injury repair.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of GDF6 and an over-expression reagent thereof in preparation of a myocardial cell protective agent.
Background
Heart failure, the terminal stage of various heart diseases, seriously threatens the health of the people in China. According to the reports 2020 on Chinese cardiovascular health and disease and the yearbook 2020 on Chinese health statistics, the prevalence rate of cardiovascular diseases in China is in a continuously rising stage, about 50% of cardiovascular disease deaths are myocardial ischemia and subsequent heart failure, and the 5-year death rate of patients with heart failure is nearly 50%. Massive cardiomyocyte death and scarring following myocardial ischemia are important causes of heart failure. The existing clinical treatment measures such as medicines, intervention, apparatuses and the like cannot solve the problem of repairing and protecting damaged myocardial tissues. With myocardial ischemia, cardiomyocytes are subjected to persistent environmental stresses including severe deficiency of oxygen or nutrients, low pH, disturbances in amino acid metabolism, etc., which cause an imbalance in cellular metabolism and redox homeostasis, the accumulation of Reactive Oxygen Species (ROS), the induction of large-scale programmed cell death (e.g., ROS initiates apoptosis by damaging mitochondria) and regulated cell death (e.g., ROS induces iron death by lethal lipid peroxidation) in cardiomyocytes. Therefore, how to promote the repair of damaged myocardial tissues and the reconstruction of cardiac functions by protecting the survival of myocardial cells is one of the major scientific problems to be solved urgently in the field of cardiovascular disease research, and a new method for protecting the survival of myocardial cells after myocardial ischemia needs to be developed urgently.
Growth differentiation factor 6 (GDF 6), one of the members of the TGF- β superfamily, belongs to the BMP family, and its amino acid sequence is highly conserved among vertebrates. GDF6 is currently well-defined in regulating skeletal and ocular development in many species, including humans. Patients with mutated GDF6 genes, such as Klippel-Feil syndrome patients, may show skeletal dysplasia, such as vertebral body fusion and joint dysplasia, in addition to ocular pathologies, including microphthalmia and age-related macular degeneration. The GDF6 gene is located on human chromosome 8, and is different from GDF11 and GDF15 which are currently researched more in the cardiovascular field, the GDF6 gene and the GDF11 and the GDF15 gene are respectively located on human chromosome 12 and chromosome 19, and the GDF6 gene, the GDF11 gene and the GDF15 gene have no similarity on nucleotide sequences and amino acid sequences of encoded proteins. Also because of this, there is no international literature on the protection of cardiomyocytes against GDF6 survival following myocardial ischemia, particularly in reducing apoptosis and iron death.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of GDF6 and an overexpression agent thereof in preparation of a myocardial cell protective agent. The invention firstly proposes that GDF6 overexpression can reduce the apoptosis and iron death of myocardial cells after myocardial ischemia and promote myocardial survival, and is expected to provide a new theoretical basis and a potential intervention target point for myocardial injury repair.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides the use of growth differentiation factor 6 in the preparation of a cardiomyocyte protective agent.
As described in the background section, growth differentiation factor 6 (GDF 6) has been well-established in the prior art for regulating skeletal and ocular development in many species, including humans, and has not been reported for its protection of cardiac myocytes. Although GDF11 and GDF15 are currently being studied more in the cardiovascular field, they are located on human chromosomes 12 and 19, respectively, while the GDF6 gene is located on human chromosome 8; and GDF6 and GDF11, GDF15 have no similarity in nucleotide sequence and amino acid sequence of encoded protein. Also based on this, no previous scholars have focused on the role of GDF6 in cardiomyocyte protection. Therefore, international reports on the protection of myocardial cell survival after myocardial ischemia by GDF6, especially the reduction of apoptosis and iron death, are not available at present. The invention firstly proposes that GDF6 overexpression can reduce the apoptosis and iron death of myocardial cells after myocardial ischemia and promote myocardial survival, and is expected to provide a new theoretical basis and a potential intervention target point for myocardial injury repair.
Further, the cardiomyocyte protective agent is a cardiomyocyte protective agent that reduces apoptosis or iron death of cardiomyocytes.
In a second aspect, the invention provides an application of a growth differentiation factor 6 overexpression reagent in preparation of a myocardial cell protective agent.
Further, the cardiomyocyte protective agent is a cardiomyocyte protective agent capable of reducing cardiomyocyte apoptosis or iron death.
Further, the overexpression reagent of the growth differentiation factor 6 is selected from lentivirus or adeno-associated virus vector inserted with growth differentiation factor 6 gene.
In a third aspect, the present invention provides a cardiomyocyte protective agent comprising growth differentiation factor 6 or an agent capable of overexpressing the growth differentiation factor 6 gene in an animal.
Preferably, the cardiomyocyte protective agent further comprises a pharmaceutically acceptable carrier and/or excipient.
Further, the myocardial cell protective agent is an injection preparation.
Still further, the injectable formulation is an intravenous drip formulation.
Compared with the prior art, the invention has the following technical effects:
(1) The invention performs GDF6 overexpression treatment on the myocardial ischemia model, shows that the GDF6 has the capability of reducing myocardial cell apoptosis and iron death after myocardial ischemia and promoting myocardial survival, and is expected to provide a new theoretical basis and a potential intervention target point for myocardial injury repair.
(2) The myocardial cell protective agent of the invention comprises two modes: (1) directly taking GDF6 as a myocardial cell protective agent; (2) an agent capable of overexpressing a GDF6 gene is used as a cardiomyocyte protective agent, thereby promoting GDF6 synthesis in vivo.
Drawings
FIG. 1 shows that after mice are subjected to an acute myocardial ischemia model, GDF 6-overexpressing lentivirus (GDF 6) is injected into ischemic heart regions of the mice oe ) Negative control lentivirus (NC) and solvent control (DMEM). Wherein: A. immunofluorescent staining patterns of heart sections 28 days after virus injection. Green is Troponin, which represents the cardiac muscle cell; red, WGA, represents the ischemic area of the heart; blue DAPI, representing nuclei. Scale =400 μm. B. Statistics of the areas of viable cardiomyocytes in the three cardiac ischemic areas. *P<0.05。
FIG. 2 shows that after mice were subjected to an acute myocardial ischemia model, GDF 6-overexpressing lentivirus (GDF 6) was injected into ischemic heart regions of the mice oe ) Negative control lentivirus (NC) and solvent control (DMEM). Wherein: A. immunofluorescent staining patterns of heart sections 28 days after virus injection. Green is Troponin, which represents the cardiac muscle cells; TUNEL in red, representing apoptotic cells; blue DAPI, representing nuclei. Scale =100 μm. B. And (3) statistics of the apoptosis of myocardial cells in the three groups of ischemic peripheral areas of the heart. *P<0.05。
FIG. 3 shows primary milk mouse cardiomyocytes transfected with GDF 6-overexpressing adenovirus (GDF 6) separately oe ) And negative control adenovirus (NC). Respectively carrying out normal oxygen culture or hypoxia-ischemia clear induction simulating in vivo hypoxia-ischemia environment for 24 hours. Wherein: A. immunofluorescence staining pattern of apoptotic cardiomyocytes. Green is Troponin, which represents the cardiac muscle cell; TUNEL in red, representing apoptotic cells; blue DAPI, representing nuclei. Scale =200 μm. B. Statistics of apoptosis in three groups of cardiomyocytes. *P<0.05。
FIG. 4 shows primary milk mouse cardiomyocytes transfected with GDF 6-overexpressing adenovirus (GDF 6) separately oe ) And negative control adenovirus (NC), cultured with complete medium or without cystine medium, respectively, for 48 hours. As a positive control, cystine-free medium was set to which 1. Mu.M ferrostatin-1 (Fer-1, iron death inhibitor) was added. Statistics of cardiomyocyte survival in three groups. *P<0.05。
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
An application of growth differentiation factor 6 in preparing myocardial cell protectant is disclosed. Further, the cardiomyocyte protective agent is a cardiomyocyte protective agent that reduces apoptosis or iron death of cardiomyocytes.
An application of the over-expression reagent of growth differentiation factor 6 in preparing the protecting agent of myocardial cells. Further, the cardiomyocyte protective agent is a cardiomyocyte protective agent that reduces apoptosis or iron death of cardiomyocytes. The over-expression reagent of the growth differentiation factor 6 is selected from lentivirus or adeno-associated virus vector inserted with growth differentiation factor 6 gene.
A cardiomyocyte protective agent comprises growth differentiation factor 6 or an agent capable of overexpressing the growth differentiation factor 6 gene, and a pharmaceutically acceptable carrier and/or excipient. Further, the myocardial cell protective agent is an injection preparation. Still further, the injectable formulation is an intravenous drip
Example 1
GDF6 overexpression virus preparation: the nucleotide sequence of GDF6 of the present invention is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO. 2. GDF6 over-expression lentivirus vector GV314 (CMV-MCS-3 FLAG-SV 40-EGFP) and GDF6 over-expression adenovirus vector GV492 (Ubi-MCS-3 FLAG-CBh-gcGFP-IRES-puromycin) used in the embodiment of the invention are all purchased from Kjekay Gene science and technology Co., ltd. SEQ ID NO.1:
atggatactcccagggtcctgctctcggccgtcttcctcatcagttttctgtgggatttgcccggtttcc
agcaggcttccatctcatcctcctcgtcgtccgccgagctgggttccaccaagggcatgcgaagccgcaa
ggaaggcaagatgcagcgggcgccgcgcgacagtgacgcgggccgggagggccaggaaccacagccgcgg
cctcaggacgaaccccgggctcagcagccccgggcgcaggagccgccaggcaggggtccgcgcgtggtgc
cccacgagtacatgctgtcaatctacaggacttactccatcgctgagaagctgggcatcaatgccagctt
tttccagtcttccaagtcggctaatacgatcaccagctttgtagacaggggactagacgatctctcgcac
actcctctccggagacagaagtatttgtttgatgtgtccatgctctcagacaaagaagagctggtgggcg
cggagctgcggctctttcgccaggcgccctcagcgccctgggggccaccagccgggccgctccacgtgca
gctcttcccttgcctttcgcccctactgctggacgcgcggaccctggacccgcagggggcgccgccggcc
ggctgggaagtcttcgacgtgtggcagggcctgcgccaccagccctggaagcagctgtgcttggagctgc
gggccgcatggggcgagctggacgccggggaggccgaggcgcgcgcgcggggaccccagcaaccgccgcc
cccggacctgcggagtctgggcttcggccggagggtgcggcctccccaggagcgggccctgctggtggta
ttcaccagatcccagcgcaagaacctgttcgcagagatgcgcgagcagctgggctcggccgaggctgcgg
gcccgggcgcgggcgccgaggggtcgtggccgccgccgtcgggcgccccggatgccaggccttggctgcc
ctcgcccggccgccggcggcggcgcacggccttcgccagtcgccatggcaagcggcacggcaagaagtcc
aggctacgctgcagcaagaagcccctgcacgtgaacttcaaggagctgggctgggacgactggattatcg
cgcccctggagtacgaggcctatcactgcgagggtgtatgcgacttcccgctgcgctcgcacctggagcc
caccaaccacgccatcatccagacgctgatgaactccatggaccccggctccaccccgcccagctgctgc
gtgcccaccaaattgactcccatcagcattctatacatcgacgcgggcaataatgtggtctacaagcagt
acgaggacatggtggtggagtcgtgcggctgcaggtag
SEQ ID NO.2
MDTPRVLLSAVFLISFLWDLPGFQQASISSSSSSAELGSTKGMRSRKEGKMQRAPRDSDAGREGQEPQPR
PQDEPRAQQPRAQEPPGRGPRVVPHEYMLSIYRTYSIAEKLGINASFFQSSKSANTITSFVDRGLDDLSH
TPLRRQKYLFDVSMLSDKEELVGAELRLFRQAPSAPWGPPAGPLHVQLFPCLSPLLLDARTLDPQGAPPA
GWEVFDVWQGLRHQPWKQLCLELRAAWGELDAGEAEARARGPQQPPPPDLRSLGFGRRVRPPQERALLVV
FTRSQRKNLFAEMREQLGSAEAAGPGAGAEGSWPPPSGAPDARPWLPSPGRRRRRTAFASRHGKRHGKKS
RLRCSKKPLHVNFKELGWDDWIIAPLEYEAYHCEGVCDFPLRSHLEPTNHAIIQTLMNSMDPGSTPPSCC
VPTKLTPISILYIDAGNNVVYKQYEDMVVESCGCR
example 2
(1) Preparation of mouse acute myocardial ischemia model: anesthetizing C57BL/6 mouse (Shanghai slake laboratory animals Co., ltd.) with chloral hydrate, intubating trachea, supporting by small animal respirator, opening chest, ligating left anterior descending coronary artery under left atrial appendage with 8-0 silk thread, determining myocardial ischemia model according to local tissue color change, and injecting GDF6 over-expressed lentivirus (GDF 6) into myocardium respectively oe ) Negative control lentivirus (NC) and solvent control (DMEM).
(2) Material taking: at 28 days post virus injection, mice were sacrificed under chloral hydrate anesthesia and hearts were collected.
(3) Staining of heart tissue: embedding and freezing heart tissues, and co-staining WGA and a Troponin antibody (Abcam, ab 47003) to evaluate myocardial survival in an ischemic region of the heart; using the TUNEL kit (Roche,In Situcell Death Detection Kit, TMR red) and Troponin antibody (Abcam, ab 47003)And (3) co-staining to evaluate the apoptosis of the myocardial cells in the peripheral area of the cardiac ischemia.
(4) As shown in FIG. 1, GDF6 oe The area ratio of the viable myocardial cells in the ischemic area of the group is 18.78 +/-3.20%, and the area ratio is remarkably increased relative to that of an NC group (9.24 +/-1.93%) and a DMEM group (8.77 +/-2.23%). As shown in FIG. 2, GDF6 oe The myocardial cell apoptosis rate of the ischemic peripheral area of the group is 8.15 +/-2.63 percent, and is obviously reduced relative to that of an NC group (18.18 +/-2.80 percent) and a DMEM group (18.03 +/-2.07 percent). The results show that local GDF6 overexpression lentivirus injection in myocardial ischemia can obviously increase the survival myocardial cell area in the ischemic area and reduce the myocardial cell apoptosis in the ischemic peripheral area.
Example 3
(1) Isolation and culture of myocardial cells in suckling mice: myocardial tissue of a newborn mouse born for 1 to 3 days is washed clean of blood clots by PBS and digested by 0.05 percent of type II collagenase and 0.05 percent of pancreatin. For 10 minutes of each digestion, the cell digestate was placed in high-glucose DMEM medium supplemented with 10% fetal calf serum to stop digestion. Repeating for multiple times until digestion is complete. The cell suspension adopts a differential adherence method to remove heart fibroblasts as much as possible so as to obtain the myocardial cells, and the high-glucose DMEM culture medium is added with 10% fetal calf serum for conventional culture.
(2) Virus transfection: and (3) carrying out adherent culture on myocardial cells of the suckling mice for 24 hours, and respectively transfecting GDF6 over-expression adenovirus and control negative adenovirus for 48 hours.
(3) Hypoxia-serum-deficient stress treatment induced apoptosis: the transfected mammary mouse myocardial cells are cultured for 24 hours under normal oxygen conditions (21% oxygen concentration, 10% fetal bovine serum is added to high-glucose DMEM medium) and 24 hours under low-sugar hypoxic-ischemic serum (0.3% oxygen concentration, low-sugar DMEM medium, serum-free).
(4) Apoptotic cardiomyocyte staining: the myocardial cells of the suckling mice are fixed by 4 percent paraformaldehyde by adopting TUNEL kit (In SituCell Death Detection Kit, tmrred, roche) and the Troponin antibody (Abcam, ab 47003) were co-stained to assess the apoptotic status of cardiomyocytes.
(5) As shown in FIG. 3, the NC group cardiomyocytes were apoptotic under hypoxic (Hypoxia) ischemia-stress conditionsThe rate is 12.68 +/-2.19%, and is obviously increased relative to the normal culture (0.09 +/-0.06%) of the normal oxygen (Normoxia), and the GDF6 is subjected to Hypoxia (Hypoxia) ischemia stress oe The apoptosis rate of the myocardial cells in the group (4.83 +/-0.56%) is remarkably reduced relative to that in the NC group, and the results show that the GDF6 overexpression can reduce the apoptosis of the myocardial cells under the condition of hypoxia and serum-lacking stress.
Example 4
(1) Isolation and culture of myocardial cells in suckling mice: the myocardial tissue of a lactating mouse born for 1-3 days is washed clean of blood clots with PBS and digested with 0.05% type II collagenase and 0.05% trypsin. For 10 minutes of each digestion, the cell digestate was placed in high-glucose DMEM medium supplemented with 10% fetal calf serum to stop digestion. Repeating the steps for multiple times until the digestion is complete. The cell suspension adopts a differential adherence method to remove heart fibroblasts as much as possible to obtain the myocardial cells, and the high-glucose DMEM culture medium is added with 10 percent fetal calf serum for conventional culture.
(2) Virus transfection: and (3) carrying out adherent culture on the myocardial cells of the suckling mice for 24 hours, and respectively transfecting GDF6 overexpression adenovirus and control negative adenovirus for 48 hours.
(3) Cystine deprivation induced iron death: the transfected mammary mouse cardiac muscle cells were cultured for 48 hours in complete medium or in cystine-free medium, respectively. Meanwhile, a cystine-free culture medium is set and 1 mu M ferrostatin-1 (Fer-1, iron death inhibitor) is added as a positive control.
(4) And (3) cell survival rate identification: myocardial Cell survival of normally cultured and cystine deprived milk mice was evaluated using CellTiter-Glo kit (Automated CellTiter-Glo Luminessent Cell Viability Assay Protocol).
(5) As shown in FIG. 4, in the absence of Cystine [ Cystine (-)]Under the culture condition, the survival rate of the myocardial cells of the NC group is 11.02 +/-1.36 percent relative to the normal culture [ Cystine (+)]Obviously reduces, but the addition of Fer-1 can improve the survival rate of the myocardial cells to 51.68 +/-1.46 percent, reverse the incidence of iron death and use GDF6 oe The survival rate of the myocardial cells in the group is improved to 25.60 +/-1.70%, and the results indicate that the GDF6 overexpression can reduce the iron death caused by cystine deprivation.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modifications, alterations and equivalent changes made to the above embodiment according to the technical spirit of the present invention still belong to the protection scope of the technical solution of the present invention.
Sequence listing
<110> Zhejiang university
<120> GDF6 and application of over-expression reagent thereof in preparation of myocardial cell protective agent
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1368
<212> DNA
<213> growth differentiation factor 6 (GDF 6)
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atggatactc ccagggtcct gctctcggcc gtcttcctca tcagttttct gtgggatttg 60
cccggtttcc agcaggcttc catctcatcc tcctcgtcgt ccgccgagct gggttccacc 120
aagggcatgc gaagccgcaa ggaaggcaag atgcagcggg cgccgcgcga cagtgacgcg 180
ggccgggagg gccaggaacc acagccgcgg cctcaggacg aaccccgggc tcagcagccc 240
cgggcgcagg agccgccagg caggggtccg cgcgtggtgc cccacgagta catgctgtca 300
atctacagga cttactccat cgctgagaag ctgggcatca atgccagctt tttccagtct 360
tccaagtcgg ctaatacgat caccagcttt gtagacaggg gactagacga tctctcgcac 420
actcctctcc ggagacagaa gtatttgttt gatgtgtcca tgctctcaga caaagaagag 480
ctggtgggcg cggagctgcg gctctttcgc caggcgccct cagcgccctg ggggccacca 540
gccgggccgc tccacgtgca gctcttccct tgcctttcgc ccctactgct ggacgcgcgg 600
accctggacc cgcagggggc gccgccggcc ggctgggaag tcttcgacgt gtggcagggc 660
ctgcgccacc agccctggaa gcagctgtgc ttggagctgc gggccgcatg gggcgagctg 720
gacgccgggg aggccgaggc gcgcgcgcgg ggaccccagc aaccgccgcc cccggacctg 780
cggagtctgg gcttcggccg gagggtgcgg cctccccagg agcgggccct gctggtggta 840
ttcaccagat cccagcgcaa gaacctgttc gcagagatgc gcgagcagct gggctcggcc 900
gaggctgcgg gcccgggcgc gggcgccgag gggtcgtggc cgccgccgtc gggcgccccg 960
gatgccaggc cttggctgcc ctcgcccggc cgccggcggc ggcgcacggc cttcgccagt 1020
cgccatggca agcggcacgg caagaagtcc aggctacgct gcagcaagaa gcccctgcac 1080
gtgaacttca aggagctggg ctgggacgac tggattatcg cgcccctgga gtacgaggcc 1140
tatcactgcg agggtgtatg cgacttcccg ctgcgctcgc acctggagcc caccaaccac 1200
gccatcatcc agacgctgat gaactccatg gaccccggct ccaccccgcc cagctgctgc 1260
gtgcccacca aattgactcc catcagcatt ctatacatcg acgcgggcaa taatgtggtc 1320
tacaagcagt acgaggacat ggtggtggag tcgtgcggct gcaggtag 1368
<210> 2
<211> 455
<212> PRT
<213> growth differentiation factor 6 (GDF 6)
<400> 2
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20 25 30
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50 55 60
Gln Glu Pro Gln Pro Arg Pro Gln Asp Glu Pro Arg Ala Gln Gln Pro
65 70 75 80
Arg Ala Gln Glu Pro Pro Gly Arg Gly Pro Arg Val Val Pro His Glu
85 90 95
Tyr Met Leu Ser Ile Tyr Arg Thr Tyr Ser Ile Ala Glu Lys Leu Gly
100 105 110
Ile Asn Ala Ser Phe Phe Gln Ser Ser Lys Ser Ala Asn Thr Ile Thr
115 120 125
Ser Phe Val Asp Arg Gly Leu Asp Asp Leu Ser His Thr Pro Leu Arg
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Arg Gln Lys Tyr Leu Phe Asp Val Ser Met Leu Ser Asp Lys Glu Glu
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Claims (3)
1. A method for protecting cardiomyocytes using growth differentiation factor 6, comprising: under in vitro conditions, cardiomyocytes were allowed to overexpress growth differentiation factor 6.
2. The method of claim 1, wherein: the method is for reducing cardiomyocyte apoptosis or iron death.
3. The method of claim 1 or 2, wherein: the over-expression of the growth differentiation factor 6 in the myocardial cells is realized by adopting an over-expression reagent of the growth differentiation factor 6; the over-expression reagent of the growth differentiation factor 6 is selected from lentivirus or adeno-associated virus vector inserted with growth differentiation factor 6 gene.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999031136A2 (en) * | 1997-12-17 | 1999-06-24 | Creative Biomolecules, Inc. | Methods for maintaining or restoring tissue-appropriate phenotype of soft tissue cells |
| CN109608551A (en) * | 2018-12-04 | 2019-04-12 | 西北大学 | A kind of HLC-BMP fusion protein and preparation method thereof |
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| US20030104977A1 (en) * | 2000-03-31 | 2003-06-05 | Ugo Ripamonti | Methods for inducing angiogenesis using morphogenic proteins and stimulatory factors |
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